CAraCAl: CAMML with the integration of chromatin accessibility.

BACKGROUND: A vital step in analyzing single-cell data is ascertaining which cell types are present in a dataset, and at what abundance. In many diseases, the proportions of varying cell types can have important implications for health and prognosis. Most approaches for cell type annotation have centered around cell typing for single-cell RNA-sequencing (scRNA-seq) and have had promising success. However, reliable methods are lacking for many other single-cell modalities such as single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), which quantifies the extent to which genes of interest in each cell are epigenetically "open" for expression. RESULTS: To leverage the informative potential of scATAC-seq data, we developed CAMML with the integration of chromatin accessibility (CAraCAl), a bioinformatic method that performs cell typing on scATAC-seq data. CAraCAl performs cell typing by scoring each cell for its enrichment of cell type-specific gene sets. These gene sets are composed of the most upregulated or downregulated genes present in each cell type according to projected gene activity. CONCLUSIONS: We found that CAraCAl does not improve performance beyond CAMML when scRNA-seq is present, but if only scATAC-seq is available, CAraCAl performs cell typing relatively successfully. As such, we also discuss best practices for cell typing and the strengths and weaknesses of various cell annotation options.

Cell type-specific interaction analysis using doublets in scRNA-seq.

Summary: Doublets are usually considered an unwanted artifact of single-cell RNA-sequencing (scRNA-seq) and are only identified in datasets for the sake of removal. However, if cells have a juxtacrine interaction with one another in situ and maintain this association through an scRNA-seq processing pipeline that only partially dissociates the tissue, these doublets can provide meaningful biological information regarding the intercellular signals and processes occurring in the analyzed tissue. This is especially true for cases such as the immune compartment of the tumor microenvironment, where the frequency and the type of immune cell juxtacrine interactions can be a prognostic indicator. We developed Cell type-specific Interaction Analysis using Doublets in scRNA-seq (CIcADA) as a pipeline for identifying and analyzing biologically meaningful doublets in scRNA-seq data. CIcADA identifies putative doublets using multi-label cell type scores and characterizes interaction dynamics through a comparison against synthetic doublets of the same cell type composition. In performing CIcADA on several scRNA-seq tumor datasets, we found that the identified doublets were consistently upregulating expression of immune response genes. Availability and implementation: An R package implementing the CIcADA method is in development and will be released on CRAN, but for now it is available at https://github.com/schiebout/CAMML.

Cell type-specific Interaction Analysis using Doublets in scRNA-seq (CIcADA).

Motivation: Doublets are usually considered an unwanted artifact of single-cell RNA-sequencing (scRNA-seq) and are only identified in datasets for the sake of removal. However, if cells have a juxtacrine attachment to one another in situ and maintain this association through an scRNA-seq processing pipeline that only partially dissociates the tissue, these doublets can provide meaningful biological information regarding the interactions and cell processes occurring in the analyzed tissue. This is especially true for cases such as the immune compartment of the tumor microenvironment, where the frequency and type of immune cell juxtacrine interactions can be a prognostic indicator. Results: We developed Cell type-specific Interaction Analysis using Doublets in scRNA-seq (CIcADA) as a pipeline for identifying and analyzing biological doublets in scRNA-seq data. CIcADA identifies putative doublets using multi-label cell type scores and characterizes interaction dynamics through a comparison against synthetic doublets of the same cell type composition. In performing CIcADA on several scRNA-seq tumor datasets, we found that the identified doublets were consistently upregulating expression of immune response genes. Contact: Courtney.T.Schiebout.GR@Dartmouth.edu , Hildreth.R.Frost@Dartmouth.edu.

CAMML with the Integration of Marker Proteins (ChIMP).

MOTIVATION: Cell typing is a critical task in the analysis of single-cell data, particularly when studying complex diseased tissues. Unfortunately, the sparsity and noise of single-cell data make accurate cell typing of individual cells difficult. To address these challenges, we previously developed the CAMML method for multi-label cell typing of single-cell RNA-sequencing (scRNA-seq) data. CAMML uses weighted gene sets to score each profiled cell for multiple potential cell types. While CAMML outperforms other scRNA-seq cell typing techniques, it only leverages transcriptomic data so cannot take advantage of newer multi-omic single-cell assays that jointly profile gene expression and protein abundance (e.g. joint scRNA-seq/CITE-seq). RESULTS: We developed the CAMML with the Integration of Marker Proteins (ChIMP) method to support multi-label cell typing of individual cells jointly profiled via scRNA-seq and CITE-seq. ChIMP combines cell type scores computed on scRNA-seq data via the CAMML approach with discretized CITE-seq measurements for cell type marker proteins. The multi-omic cell type scores generated by ChIMP allow researchers to more precisely and conservatively cell type joint scRNA-seq/CITE-seq data. AVAILABILITY AND IMPLEMENTATION: An implementation of this work is available on CRAN at https://cran.r-project.org/web/packages/CAMML/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The evolutionary history of small RNAs in Solanaceae.

The Solanaceae or "nightshade" family is an economically important group with remarkable diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family's small RNA (sRNA) genomic landscape, we downloaded over 255 publicly available sRNA data sets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major sRNA classes: (1) microRNAs (miRNAs), typically 20- to 22-nucleotide (nt) RNAs generated from a hairpin precursor and functioning in gene silencing and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs and trans-acting siRNAs generated via miRNA-directed cleavage of a polymerase II-derived RNA precursor. Our analyses described thousands of sRNA loci, including poorly understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these sRNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar sRNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities.

Superkine IL-2 and IL-33 Armored CAR T Cells Reshape the Tumor Microenvironment and Reduce Growth of Multiple Solid Tumors.

Chimeric-antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against hematologic tumors. Yet, CAR T-cell therapy has had little success against solid tumors due to obstacles presented by the tumor microenvironment (TME) of these cancers. Here, we show that CAR T cells armored with the engineered IL-2 superkine Super2 and IL-33 were able to promote tumor control as a single-agent therapy. IFNγ and perforin were dispensable for the effects of Super2- and IL-33-armored CAR T cells. Super2 and IL-33 synergized to shift leukocyte proportions in the TME and to recruit and activate a broad repertoire of endogenous innate and adaptive immune cells including tumor-specific T cells. However, depletion of CD8+ T cells or NK cells did not disrupt tumor control, suggesting that broad immune activation compensated for loss of individual cell subsets. Thus, we have shown that Super2 and IL-33 CAR T cells can promote antitumor immunity in multiple solid tumor models and can potentially overcome antigen loss, highlighting the potential of this universal CAR T-cell platform for the treatment of solid tumors.

CAMML: Multi-Label Immune Cell-Typing and Stemness Analysis for Single-Cell RNA-sequencing.

Inferring the cell types in single-cell RNA-sequencing (scRNA-seq) data is of particular importance for understanding the potential cellular mechanisms and phenotypes occurring in complex tissues, such as the tumor-immune microenvironment (TME). The sparsity and noise of scRNA-seq data, combined with the fact that immune cell types often occur on a continuum, make cell typing of TME scRNA-seq data a significant challenge. Several single-label cell typing methods have been put forth to address the limitations of noise and sparsity, but accounting for the often overlapped spectrum of cell types in the immune TME remains an obstacle. To address this, we developed a new scRNA-seq cell-typing method, Cell-typing using variance Adjusted Mahalanobis distances with Multi-Labeling (CAMML). CAMML leverages cell type-specific weighted gene sets to score every cell in a dataset for every potential cell type. This allows cells to be labelled either by their highest scoring cell type as a single label classification or based on a score cut-off to give multi-label classification. For single-label cell typing, CAMML performance is comparable to existing cell typing methods, SingleR and Garnett. For scenarios where cells may exhibit features of multiple cell types (e.g., undifferentiated cells), the multi-label classification supported by CAMML offers important benefits relative to the current state-of-the-art methods. By integrating data across studies, omics platforms, and species, CAMML serves as a robust and adaptable method for overcoming the challenges of scRNA-seq analysis.

iCAT: diagnostic assessment tool of immunological history using high-throughput T-cell receptor sequencing.

The pathogen exposure history of an individual is recorded in their T-cell repertoire and can be accessed through the study of T-cell receptors (TCRs) if the tools to identify them were available. For each T-cell, the TCR loci undergoes genetic rearrangement that creates a unique DNA sequence. In theory these unique sequences can be used as biomarkers for tracking T-cell responses and cataloging immunological history. We developed the immune Cell Analysis Tool (iCAT), an R software package that analyzes TCR sequencing data from exposed (positive) and unexposed (negative) samples to identify TCR sequences statistically associated with positive samples. The presence and absence of associated sequences in samples trains a classifier to diagnose pathogen-specific exposure. We demonstrate the high accuracy of iCAT by testing on three TCR sequencing datasets. First, iCAT successfully diagnosed smallpox vaccinated versus naïve samples in an independent cohort of mice with 95% accuracy. Second, iCAT displayed 100% accuracy classifying naïve and monkeypox vaccinated mice.  Finally, we demonstrate the use of iCAT on human samples before and after exposure to SARS-CoV-2, the virus behind the COVID-19 global pandemic. We were able to correctly classify the exposed samples with perfect accuracy. These experimental results show that iCAT capitalizes on the power of TCR sequencing to simplify infection diagnostics. iCAT provides the option of a graphical, user-friendly interface on top of usual R interface allowing it to reach a wider audience.

Diagnostic differentiation of Zika and dengue virus exposure by analyzing T cell receptor sequences from peripheral blood of infected HLA-A2 transgenic mice.

Zika virus (ZIKV) is a significant global health threat due to its potential for rapid emergence and association with severe congenital malformations during infection in pregnancy. Despite the urgent need, accurate diagnosis of ZIKV infection is still a major hurdle that must be overcome. Contributing to the inaccuracy of most serologically-based diagnostic assays for ZIKV, is the substantial geographic and antigenic overlap with other flaviviruses, including the four serotypes of dengue virus (DENV). Within this study, we have utilized a novel T cell receptor (TCR) sequencing platform to distinguish between ZIKV and DENV infections. Using high-throughput TCR sequencing of lymphocytes isolated from DENV and ZIKV infected mice, we were able to develop an algorithm which could identify virus-associated TCR sequences uniquely associated with either a prior ZIKV or DENV infection in mice. Using this algorithm, we were then able to separate mice that had been exposed to ZIKV or DENV infection with 97% accuracy. Overall this study serves as a proof-of-principle that T cell receptor sequencing can be used as a diagnostic tool capable of distinguishing between closely related viruses. Our results demonstrate the potential for this innovative platform to be used to accurately diagnose Zika virus infection and potentially the next emerging pathogen(s).

Identifying and Tracking Low-Frequency Virus-Specific TCR Clonotypes Using High-Throughput Sequencing.

Tracking antigen-specific T cell responses over time within individuals is difficult because of lack of knowledge of antigen-specific TCR sequences, limitations in sample size, and assay sensitivities. We hypothesized that analyses of high-throughput sequencing of TCR clonotypes could provide functional readouts of individuals' immunological histories. Using high-throughput TCR sequencing, we develop a database of TCRß sequences from large cohorts of mice before (naive) and after smallpox vaccination. We computationally identify 315 vaccine-associated TCR sequences (VATS) that are used to train a diagnostic classifier that distinguishes naive from vaccinated samples in mice up to 9 months post-vaccination with >99% accuracy. We determine that the VATS library contains virus-responsive TCRs by in vitro expansion assays and virus-specific tetramer sorting. These data outline a platform for advancing our capabilities to identify pathogen-specific TCR sequences, which can be used to identify and quantitate low-frequency pathogen-specific TCR sequences in circulation over time with exceptional sensitivity.